PROJECT SUMMARY Matrix stiffening is a defining feature of lung and liver fibrosis. While traditionally viewed as an endpoint, matrix stiffening is now recognized to develop early during fibrosis initiation, and to contribute prominently to disease progression through mechano-activation of myofibroblasts derived from lung fibroblasts (LFs) or hepatic stellate cell (HSCs). Recent studies by our group and others have identified the transcriptional effectors YAP and TAZ as key mediators of LF and HSC mechano-activation, demonstrating that a common molecular mechanism underlies myofibroblast activation in these two organs. Here we propose to dramatically expand our focus on YAP and TAZ not just as responders to matrix stiffness, but as central effectors that initiate, amplify, and maintain the matrix stiffening driven by LFs and HSCs, and thus highlight their potential as targets for therapy directed at matrix stiffening itself. In our first aim we will elucidate the mechanistic roles that YAP and TAZ play in initiation, maintenance and propagation of LF- and HSC-mediated lung and liver stiffening, using xenografting to study cell-mediated stiffening of the intact organ, and cellular remodeling of a model ECM to elucidate specific contributions of YAP and TAZ to matrix stiffening via extracellular matrix contraction, synthesis, crosslinking and breakdown. Recognizing that YAP and TAZ play prominent roles in a multitude of cell types and contexts, in our second aim we seek to identify mechanisms by which LF/HSC-specific pro- fibrotic activation of YAP and TAZ can be selectively ablated. We focus on a mechanistic approach to target G protein coupled receptor signaling through a Gs/cAMP pathway as a cell-specific mechanism to inactivate YAP and TAZ and ablate LF and HSC activation even in the face of pro-fibrotic stiff matrix conditions. Invasive atomic force microscopy (AFM) and non-invasive magnetic resonance elastography (MRE) approaches will be employed to test the efficacy of targeting these pathways in preventing or reversing matrix stiffening in pre- clinical models of lung and liver fibrosis. The proposed studies will elucidate novel molecular mechanisms linking cellular activation to matrix stiffening in vitro and in vivo, and identify new approaches to ablate LF and HSC activation in a cell-specific manner, important first steps toward new therapies targeting matrix stiffness and fibrosis. Validation of MRE as a metric to evaluate therapeutic targeting and clinical progression of matrix stiffening will position us to translate this approach to the clinic for patients with fibrotic diseases of the lung and liver.